The immune response relies on leukocyte trafficking and immune surveillance as one of the underpinnings of host defense. Not only does this immune surveillance allow leukocytes to recirculate through lymphoid tissues normally, but also permits rapid leukocyte recruitment and extravasation to adjacent tissues at sites of inflammation. The .alpha.4.beta.1 (CD49d/CD29, VLA-4) cell adhesion receptor is an active participant in these leukocyte trafficking functions [Hemler, Ann. Rev. Immunol., 8:365-400 (1990); Hemler et al., Immunol. Rev., 114:45-65 (1990)].
The VLA-4 integrin heterodimer was discovered independently by three research groups and identified as a surface antigen on lymphocytes [Sanchez-Madrid et al., Eur. J. Immunol., 16:1343-1349 (1986); Clayberger et al., J. Immunol., 138:1510-1514 (1987); Hemler et al., J. Biol. Chem., 262:11478-11485 (1987)]. Within the integrin family, VLA-4 is unique on several counts: (i) in contrast to related members of the .beta.1 subfamily, VLA-4 is predominantly expressed on cells of the hematopoietic lineage [Hemler, Ann. Rev. Immunol., 8:365-400 (1990)], and is functionally involved in cell-cell, as well as cell-extracellular matrix (ECM) adhesive interactions [Hemler, Ann. Rev. Immunol., 8:365-400 (1990)]; (ii) despite sequence homology with other integrin .alpha. subunits, the .alpha.4 subunit stands apart from the two major structural clusters of a subunits because .alpha.4 lacks an inserted I-domain, and does not undergo post-translational cleavage near the transmembrane region [Hemler, Ann. Rev. Immunol., 8:365-400 (1990); Hynes, Cell, 69:11-25 (1992)]; and (iii) .alpha.4 contains a trypsin-like cleavage site that results in cell type-specific surface expression of at least two different structural variants termed .alpha.4-150 and .alpha.4-80/70 [Pulido et al., FEBS Lett., 294:121-124 (1991); Teixido et al., J. Biol. Chem., 267:1786-1791 (1992); Rubio et al., Eur. J. Immunol., 22:1099-1102 (1992)].
The VLA-4 integrin appears to be one of the earliest adhesion receptors found on CD34-expressing hematopoietic stem cells [Teixido et al., J. Clin. Invest., 90:358-367 (1992)]. However, VLA-4 is expressed only on mature T and B lymphocytes, natural killer (NK) cells, monocytes, basophils and eosinophils, but not on erythrocytes, platelets and neutrophils [Hemler, Ann. Rev. Immunol., 8:365-400 (1990); Gismondi et al., J. Immunol., 146:384-392 (1991); Walsh et al., J. Immunol., 146:3419-3423 (1991); Bochner et al., J. Exp. Med., 173:1553-1556 (1992); Dobrina et al., J. Clin. Invest., 88:20-26 (1991); Weller et al., Proc. Natl. Acad. Sci. USA, 88:7430-7433 (1991)].
To date, most adhesion functions mediated by VLA-4 can be explained by a direct molecular interaction between the VLA-4 integrin and either of two separate counterreceptor structures, namely, the cytokine-inducible vascular cell adhesion molecule-1 (VCAM-1) [Elices et al., Cell, 60:577-584 (1990); Rice et al., J. Exp. Med., 171:1369-1374 (1990); Schwartz et al., J. Clin. Invest., 85:2019-2022 (1990); Carlos et al., Blood, 76:965-970 (1990)], and a subset of the ubiquitous ECM protein fibronectin [Wayner et al., J. Cell Biol., 109:1321-1330 (1989); Guan et al., Cell, 60:53-61 (1990); Ferreira et al., J. Exp. Med., 171:351-356 (1990); Elices et al., Cell, 60:577-584 (1990)].
VCAM-1 is a member of the immunoglobulin (Ig) gene superfamily [Osborn et al., Cell, 59:1203-1211 (1989); Rice et al., Science, 246:1303-1306 (1989)] that is expressed predominantly in vascular endothelium in response to pro-inflammatory cytokines such as IL-1, TNF.alpha., and IL-4 [Osborn et al., Cell, 59:1203-1211 (1989); Rice et al., Science, 246:1303-1306 (1989); Thornhill et al., J. Immunol., 145:865-872 (1990); Masinovsky et al., J. Immunol., 145:2886-2895 (1990); Thornhill et al., J. Immunol., 146:592-598 (1991); Schleimer et al., J. Immunol., 148:1086-1092 (1992); Birdsall et al., J. Immunol., 148:2717-2723 (1992); Swerlick et al., J. Immunol., 149:798-705 (1992); Briscoe et al., J. Immunol., 149:2954-2960 (1992)]. The VLA-4 binding sites on VCAM-1 have been mapped to the outermost N-terminal (first) Ig-like region of the 6-Ig-like domain VCAM-1 isoform [Taichman et al., Cell Regul., 2:347-355 (1991); Vonderheide et al., J. Exp. Med., 175:1433-1442 (1992); Osborn et al., J. Exp. Med., 176:99-107 (1992)], and the first and fourth N-terminal Ig-like regions of the 7-Ig-like domain VCAM-1 isoform [Vonderheide et al., J. Exp. Med., 175:1433-1442 (1992); Osborn et al., J. Exp. Med., 176:99-107 (1992)]. Discrete amino acid sequences within the two separate Ig-like domains in VCAM-1 recognized by the VLA-4 integrin remain to be defined.
In contrast, a high affinity peptide recognition sequence for VLA-4 within fibronectin (FN) has been identified [Wayner et al., J. Cell. Biol., 109:1321-1330 (1989); Ferreira et al., J. Exp. Med., 171:351-356 (1990); Guan et al., Cell, 60:53-61 (1990); Mould et al., J. Biol. Chem., 265:4020-4024 (1990); Garcia-Pardo et al., J. Immunol., 144:3361-3366 (1990); Komoriya et al., J. Biol. Chem., 266:15075-15079 (1991)]. That sequence comprises a 25-amino acid residue stretch, termed CS-1 [Humphries et al., J. Cell Biol., 103:2637-2647 (1986); Humphries et al., J. Biol. Chem., 262:6886-6892 (1987)].
The FN gene contains three separate exons termed EIIIA, EIIIB and V or IIICS, which are subject to alternative splicing [Hynes, "Fibronectin", Springer-Verlag, New York (1990)]. The presence of additional acceptor and donor splice signals within the IIICS region permits generation of increased diversity in FN by virtue of multiple IIICS polypeptide variants, for instance, five in human FN [Vibe-Pedersen et al., FEBS Lett., 207:287-291 (1987); Hershberger et al., Mol. Cell. Biol., 10:662-671 (1990)]. Consequently, only a subset of these molecular variants expresses the 25-amino acid CS-1 sequence recognized by VLA-4 [Wayner et al., J. Cell. Biol., 109:1321-1330 (1989); Guan et al., Cell, 60:53-61 (1990)].
A minimal essential sequence for specific VLA-4 recognition of CS-1 has been identified as the tripeptide Leu-Asp-Val (LDV) [Komoriya et al., J. Biol. Chem., 266:15075-15079 (1991); Wayner et al., J. Cell. Biol., 116:489-497 (1992); Wayner WO 91/03252 published Mar. 21, 1991; Wayner WO 93/12809 published Jul. 8, 1993; and Humphries WO 92/13887, published Aug. 20, 1992] albeit VLA-4 binds to LDV with at least two orders of magnitude lower affinity than to the native CS-1 25-mer. Nowlin et al., J. Biol. Chem., 268(1):20352-20359 (1993) recently described a cystine-linked cyclic pentapeptide said to inhibit binding by both the Arg-Gly-Asp and CS-1 regions of fibronectin.
VLA-4 shares with other members of the .beta.1 integrin subfamily the ability to promote binding and penetration of microbial pathogens into mammalian cells. Thus, specific interactions of .beta.1 integrins with the bacterial protein invasin [Isberg et al., Cell, 60:861-871 (1990); Ennis et al., J. Exp. Med., 177:207-212 (1993)], as well as the protozoan Trypanosoma cruzi [Fernandez et al., Eur. J. Immunol., 23:552-557 (1993)] have been described.
A multitude of in vitro studies suggest interactions of VLA-4 with its two known ligands, VCAM-1 and CS-1 FN, have profound biological significance. For instance, VLA-4 binding to VCAM-1 has been demonstrated in adhesion to cytokine-stimulated vascular endothelium by lymphocytes [Elices et al., Cell, 60:577-584 (1990); Rice et al., J. Exp. Med., 171:1369-1374 (1990); Schwartz et al., J. Clin. Invest., 85:2019-2022 (1990); Carlos et al., Blood, 76:965-970 (1990); Shimizu et al., J. Cell Biol., 113:1203-1212 (1991)], monocytes [Carlos et al., Blood, 77:2266-2271 (1991); Jonjic et al., J. Immunol., 148:2080-2083 (1992)], natural killer (NK) cells [Allavena et al., J. Exp. Med., 173:439-448 (1991)], and eosinophils [Walsh et al., J. Immunol., 146:3419-3423 (1991); Bochner et al., J. Exp. Med., 173:1553-1556 (1992); Dobrina et al., J. Clin. Invest., 88:20-26 (1991); Weller et al., Proc. Natl. Acad. Sci. USA, 88:7430-7433 (1991)]. Because of its involvement in mediating leukocyte-endothelial attachment, VLA-4/VCAM-1 interactions are considered key in inflammation.
The VLA-4/CS-1 interaction, in turn, has been widely documented in hematopoiesis where adhesive interactions between hematopoietic progenitors expressing VLA-4 [Hemler et al., Immunol. Rev., 114:45-65 (1990); Williams et al., Nature, 352:438-441 (1991); Roldan et al., J. Exp. Med., 175:1739-1747 (1992); Sawada et al., J. Immunol., 149:3517-3524 (1992); Wadsworth et al., J. Immunol., 150:847-857 (1993)] and their ECM microenvironment play a critical role in precursor maturation and differentiation. Thus, CS-1 peptides have been shown to inhibit (i) attachment of murine hematopoietic stem cells to ECM derived from bone marrow stroma [Williams et al., Nature, 352:438-441 (1991)], (ii) immunoglobulin secretion by bone marrow-derived B cell progenitors [Roldan et al., J. Exp. Med., 175:1739-1747 (1992)], (iii) bursal and postbursal development of chicken B cells [Palojoki et al., Eur. J. Immunol., 23:721-726 (1993)], and (iv) thymocyte adhesion and differentiation induced by thymic stromal cell monolayers [Utsumi et al., Proc. Natl. Acad. Sci. USA, 88:5685-5689 (1991); Sawada et al., J. Immunol., 149:3517-3524 (1992)]. VLA-4/CS-1 may also be involved in embryonic development, because CS-1 peptides have been shown to interfere with migration of avian neural crest cells [Dufour et al., EMBO J., 7:2661-2671 (1988)].
In addition to VCAM-1, FN and CS-1 have also been implicated in the pathology of rheumatoid arthritis (RA) [Laffon et al., J. Clin. Invest., 88:546-552 (1992)]. A role for the CS-1 splicing variant of FN has been established in mediating migration of inflammatory cells such as eosinophils across endothelial cell monolayers of VLA-4-expressing leukocytes [Kuijpers et al., J. Exp. Med., 178:279-284 (1993)].
The vast body of work suggesting that VLA-4 plays a role in leukocyte trafficking and inflammation has been largely confirmed by in vivo studies using anti-VLA-4 antibodies in various animal models. Essentially, the skin, brain, kidney, lung and gut are targets of a wide variety of VLA-4-dependent inflammatory reactions mostly resulting from recruitment of mononuclear leukocytes and eosinophils.
More specifically, these in vivo studies are as follows: contact hypersensitivity (CH) and delayed type hypersensitivity (DTH) in the mouse and rat [Ferguson et al., Proc. Natl. Acad. Sci. USA, 88:8072-8076 (1991); Issekutz, Cell Immunol., 138:300-312 (1991); Issekutz, J. Immunol., 147:4178-4184 (1991); Elices et al., Clin. Exp. Rheumatol., 11:S77-80 (1993); Ferguson et al., Proc. Natl. Acad. Sci. USA, 88:8072-8076 (1991); Chisholm, et al., Eur. J. Immunol., 23:682-688 (1993)]; experimental autoimmune encephalomyelitis (EAE) in the mouse and rat [Yednock et al., Nature, 356:63-66 (1992); Baron et al., J. Exp. Med., 177:57-68 (1993)]; nephrotoxic nephritis in the rat [Mulligan et al., J. Clin. Invest., 91:577-587 (1993)]; passive cutaneous anaphylaxis in the guinea pig [Weg et al., J. Exp. Med., 177:561-566 (1993)]; immune complex-induced lung injury in the rat [Mulligan et al., J. Immunol., 150:2401-2406 (1993); Mulligan et al., J. Immunol., 150:2407-2417 (1993)], spontaneous colitis in the monkey [Poldolsky et al., J. Clin. Invest., 92:372-380 (1993)] and asthma in sheep [Lobb, WO 92/13798 published Jul. 22, 1993].
Thus, a preliminary conclusion from in vivo results is that VLA-4 contributes to inflammatory responses that emulate chronic conditions in humans. In an in vivo model of murine contact hypersensitivity, the CS-1 peptide partially inhibited recruitment of T lymphocytes to skin inflammatory sites [Ferguson et al., Proc. Natl. Acad. Sci. USA, 88:8072-8076 (1991)]. Because the Arg-Gly-Asp peptide from the cell adhesion domain of FN was also inhibitory in this animal model, the authors concluded that emigration of immune T cells to sites of antigenic challenge in the tissue could be facilitated by the interaction of leukocyte integrins with ECM proteins such as FN [Ferguson et al., Proc. Natl. Acad. Sci. USA, 88:8072-8076 (1991)].
In a more recent study, Elices and coworkers [Elices et al., Clin. Exp. Rheumatol., 11:S77-80 (1993)] were unable to reproduce inhibition of contact hypersensitivity with the native CS-1 peptide. Instead, they found that the CS-1 peptide was rapidly cleared from blood circulation by proteolytic degradation.
The role of VIA-4 and the CS-1 peptide in various chronic and acute immunoinflammatory disease states having been established, it would be of importance if compounds could be found that inhibit the VLA-4-lymphocyte interaction and were other than anti-VLA-4 antibodies that can themselves induce an immune response on repeated administration or the CS-1 peptide that is large and costly to make, and also is subject to rapid degradation. The disclosure that follows describes such small molecules that are more potent than is CS-1 itself.